High-Sensitivity Measurement of Free-Protein Concentration using Optical Tweezers

摘要

We have applied optical tweezers to measure the free-protein concentration of a microscopic sample in the nM range by measuring the optical tweezer laser power at which protein coated beads are ruptured from an antibody coated coverslip surface. We used silica beads that were covalently coated with a target protein and a glass coverslip coated with antibodies specific against the target protein, causing the coated beads to stick to the surface. The unknown unlabelled target protein concentration was added, which then competed with the bead-bound target protein for antibody binding sites on the coverslip surface. In this way the number of bead-surface bonds were modulated by the free protein concentration in solution affecting the threshold laser power necessary to rupture the bead from the surface. An optical tweezer was used to probe the number of bead-surface bonds by measuring the threshold power required to pull the bead away from the surface. We positioned an optical tweezer (1064 nm) slightly above the bead and linearly ramped the laser power until the bead ruptured from the surface. The power at which this occurred was used to determine the free protein concentration. Our measured calibration curve of threshold power versus free protein concentration was fitted to a single binding site equilibrium model which yielded an estimate for the equilibrium dissociation coefficient that is comparable to literature values.